Bone substitute and method for the preparation thereof

ABSTRACT

The invention relates to a material that can be used as a bone substitute and to a method for the preparation thereof. The material comprises an organic phase (I) comprising striated collagen fibrils constituted of collagen I triple helices, said fibrils being organized over a large distance according to a 3D geometry associating aligned domains and cholesteric domains, and also isotropic domains where they are not organized; and a mineral phase (II) comprising apatite crystals having a hexagonal crystalline structure, space group 6/m, comprising at least calcium ions and phosphate ions; the axis c of said apatite crystals of the mineral phase is coaligned with the longitudinal axis of the striated collagen fibrils of the organic phase and the collagen content in the material is at least 75 mg/cm 3 . The method for preparing the material comprises preparing an initial acidic aqueous solution of collagen which is a precursor for the organic phase (I), and at least one aqueous solution of precursors for the mineral phase (II) and precipitating the collagen by increasing the pH to a value of at least 7. According to this method: the concentration of collagen in the acidic aqueous solution is at least 75 mg/ml and remains constant during said increase in pH; the mineral phase precursors comprise at least one calcium salt and at least one phosphate salt; the precipitation of the mineral phase (II) is carried out by bringing the mineral phase precursor solution into contact with the organic phase (I), said bringing into contact being carried out before or after the precipitation of said organic phase (I).

The present invention relates to a bone substitute and to a method forthe preparation thereof.

Bone is a hybrid material constituted mainly of cells, collagen type Iwhich constitutes an organic protein network, and a mineral phaseconsisting of hydroxyapatite crystals of nanometric size. Thislarge-scale organic/mineral association in three dimensions gives thebone tissue both elasticity and hardness, allowing it to withstand theforces which are applied thereto. Bone is therefore hard, dense and verystrong.

Weiner & Wagner (Ann. Rev. Mater. Sci. 28, 271-298, 1998) have proposeda description of a hierarchical organization on various scales which canbe broken down into seven levels described as follows and which areillustrated by the appended FIG. 1:

-   -   level 1 (FIG. 1 a): the two major constituent basic components        of bone, i.e. the hydroxyapatite platelets and the striated        collagen fibrils, constitute the first hierarchical level of        organization. This is the lowest level of organization, on the        nanometric scale. The apatite phase is in particular        characterized by the presence of characteristic inter-reticular        planes such as (002) and (300). The apatite crystals, at this        level of organization, do not have a particular orientation. The        collagen fibrils are characterized by a periodic striation,        which is visible by electron microscopy, and which results from        the assembly of the collagen I molecules, inducing a periodic        shift of 67 nm;    -   level 2 (FIG. 1 b): the coalignment of the hydroxyapatite        platelets according to their axis c, along the main axis of the        striated collagen fibrils, constitutes the second level, i.e.        the inter-reticular planes (002) of the apatite are oriented        perpendicular to the main axis of the fibrils and, therefore,        according to the axial periodicity (i.e. according to the        striations) of the collagen fibrils (striation=67 nm). The term        mineralized collagen fibrils is used (width 100 to 300        nanometers). Level 2 is also on the nanometric scale;    -   level 3 (FIG. 1 c): several mineralized collagen fibrils are        assembled side by side in parallel bundles, forming a        mineralized collagen fiber (width 1 to 3 micrometers). The        micrometric scale is reached at organization level 3;    -   level 4 (FIG. 1 d): it is complex since the mineralized collagen        fibrils or fibers can organize in three dimensions.        Specifically, at this level, it is possible to distinguish the        coexistence of domains where the fibrils/fibers are aligned in a        preferential direction over a large distance and/or form arched        structures characteristic of their stack according to a        “cholesteric” geometry. Domains where the fibrils/fibers do not        organize (“isotropic” domains) are also distinguished. The scale        varies from micrometric to millimetric;    -   level 5 (FIG. 1 e): the compact bone has an arrangement of        parallel cylindrical structures of millimetric size, denoted        “osteons”. In section, these osteons appear to consist of        concentric collagen lamellae;    -   level 6 (FIG. 1 f): the cell-rich central part of the long bones        is called “spongy bone”. In this bone, bone lamellae form a        macroporous network of thin and irregular rows. The compact        bone/spongy bone combination constitutes organization level 6.        The scale is more than one millimeter;    -   level 7 (FIG. 1 g): the final level is quite simply the whole        bone.

Several classes of synthetic materials (denoted “implant materials”) oris natural materials (denoted “grafts”) are proposed in the prior art.Implant materials are generally bioinert, i.e. simply tolerated by theorganism, or biocompatible, i.e. they integrate perfectly into the hostorganism. A graft is a bone tissue taken from the person for whom it isintended (autograft) or from a third person (allograft), and it isgenerally osteoconductive, i.e. it is capable of guiding bone regrowth.

An osteoinductive bone substitute, i.e. one which is capable of inducingbone reconstruction, nevertheless constitutes an ideal substitute. Thedevelopment of such a material is complex. Putting into place such amaterial requires the use of constituents which have a specificcrystalline phase and chemical nature in order to optimize perfectintegration thereof in a human or animal body, and to thus avoidrejection. Its three-dimensional organization must be reconstituted inorder to provide, firstly, the mechanical properties and, secondly, aporosity suitable for the colonization of said substitute by the hosttissue. The access to the organization of the organic bone network (20%by mass), and also the association thereof with the mineral phase (70%by mass) in the tissue are very difficult to reproduce in vitro.

Many studies have been carried out with a view to synthesizing bonesubstitutes, and in particular studies relating to collagenmineralization. The mineralization of turkey bone tendon collagen hasbeen studied by W. Traub et al. [Proc. Natl. Acad. Sci. USA 1989, 86,9822-9826], but the material obtained does not display an organizationanalogous to that of bone. Other tests have been carried out withpurified collagen in vitro, but the conditions of strong dilution underwhich the tests were carried out did not make it possible to obtain amaterial having the bone density and the three-dimensional collagenorganization that are found in living bone tissues [cf. D. Lickorish elal. (J. Biomed. Mat. Res. 2004, 68A, 19-27); S. Yunoki et al. (Mat.Lett., 2006, 60, 999-1002); D. A. Wahl et al. (Eur. Cell. Mat. 5 2006,11, 43-56)].

The crystallization of calcite CaCO₃ from a solution of CaCl₂ under anammonia atmosphere generated by the thermal decomposition at ambienttemperature of a powder of (NH₄)CO₃ has been described by L. Addadi etal. (Proc. Natl. Acad. Sci. USA 1987, 84, 2732-2736).

It is also known practice to precipitate collagen from an acid solutionby increasing the pH. R. L. Ehrman et al. (J. Nat. Cancer Inst. 1956,16, 1375-1403) describe a method in which a solution of collagen inacetic acid is brought into contact with NH₃ vapors. It transforms intoa gel containing fine grains. The structure of the material obtained isnot described.

M. M. Giraud-Guille et al. (J. Mol. Biol. 1995, 251, 197-202) and (J.Mol. Biol. 1992, 224, 861-873) describe the “liquid crystal” structureobtained using a concentrated solution of collagen and also the sol-geltransition obtained by raising the pH from acidic to basic.

G. Mosser, M. M. Giraud-Guille el al. (Matrix Biol. 2006, 25, 3-13) 20describe a method in which an acidic solution of collagen (5 mg/mL) isgradually concentrated in glass microchambers in order to obtain afar-reaching helicoidal organization of the collagen molecules and alsoa concentration gradient. The solution is then brought into contact withammonia vapors, in order to form collagen fibrils and to stabilize theorganization put in place in the liquid phase.

B. A. Harley et al. (Biomaterials 2006, 27, 866-874) describe theproduction of a structured matrix of collagen also containing aglucosaminoglycan. Collagen microfibrils are homogeneously mixed withchondroitin sulfate at 4° C. The solution is then centrifuged in a mold,ultra-rapidly frozen, freeze-dried, and then crosslinked at 105° C.under a vacuum of 50 mTorr for 24 hours. The fibrillar nature of thecollagen is not described.

C. Guo el al. (Biomaterials 2007, 28, 1105-1114) describe the use ofmagnetic beads for aligning a solution of collagen fibrils. A collagensolution prepared in a phosphate buffer at concentrations of 2.5 mg/ml,maintained at 4° C., is brought into contact with the magnetic beads.The same samples are also prepared in the presence of cells at a finalcollagen concentration of 1.2 mg/ml. In both cases, the samples areplaced in a magnetic field of less than 1G during the induction offibrillogenesis produced by an increase in temperature to 37° C. A CO₂atmosphere is also used when cells are integrated into the matrix. Thematrices are very loose and the fibrillar nature of the collagen is notmentioned. M. J. Olsza el al. (Calcif. Tissue Int. 2003, 72, 583-591)describe the calcification of a collagen sponge in the presence orabsence of a polymer of the poly(aspartic acid) type. The collagensponge is constituted of collagen type I obtained from bovine tendon.The mineral is calcium carbonate and not calcium phosphate, no apatitephase is therefore obtained. The presence of striated fibers is notdemonstrated and the in collagen fibers are not oriented. J. H. Bradt etal. (Chem. Mater. 1999, 11, 2694-2701) describe a method in which twosolutions are prepared at 4° C., the first being a solution of collagen(calf dermis collagen type I) at 1 mg/mL acidified with HCl andcontaining CaCl₂, and the second being a buffer solution containingphosphate ions. The phosphate solution is then mixed with the collagensolution, making it is possible to achieve a pH of 6.8, and the wholemixture is heated to 30° C. Coprecipitation gives a mixture of phasescontaining calcium phosphate, hydroxyapatite and octacalcium phosphate.In addition, the collagen fibers are isolated nonoriented fibers and donot constitute a dense matrix. N. Gehrke, N. Nassif et al. (Chem. Mater.2005, 17, 6514-6516) describe the remineralization, with calciumcarbonate, in the presence or absence of a polymer of the poly(asparticacid) type, of the organic network of previously demineralizedmother-of-pearl.

None of the synthesis methods known to date makes it possible to obtaina bone substitute which reproduces level 4 of three-dimensionalorganization of collagen associated with a mineral phase of apatitecrystals which is observed in natural bone.

The objective of the present invention is to provide a syntheticmaterial which can be used as a biocompatible bone substitute having astructure very close to the structure of living bone (level 4), and alsoa method for preparation thereof.

The synthetic material according to the present invention comprises anorganic phase (I) and a mineral phase (II).

The organic phase (I) comprises striated collagen fibrils constituted ofcollagen I triple helices and in which the periodicity of the striationsis approximately 67 nm, said fibrils being organized over a largedistance according to a 3D geometry associating aligned domains andcholesteric domains, and also isotropic domains where they are notorganized.

The mineral phase (II) comprises apatite crystals having a hexagonalcrystalline structure, space group 6/m, said crystals comprising atleast calcium ions and at least phosphate ions.

In the material in accordance with the invention, the axis c of theapatite crystals of the mineral phase is coaligned with the longitudinalaxis of the striated collagen fibrils of the organic phase.

The collagen content in said material is at least 75 mg/cm³.

In said material, the order of magnitude of the various domains(cholesteric, alignment, isotropic) is about fifty micronsapproximately.

According to one particular embodiment, the mineral phase consists ofpure hydroxyapatite crystals. For the purpose of the present invention,the term “pure hydroxyapatite” is intended to mean a hydroxyapatite freeof other crystalline phosphate phases, such as brushite.

In one particular embodiment of the invention, the mineral phaseconsists of crystals of stoichiometric hydroxyapatite of formula (I)below:

Ca₁₀(PO₄)₆(OH)₂   (I)

According to one particular embodiment of the invention, the Ca/P atomicratio of the crystals of hydroxyapatite of formula (I) is 1.67.

According to another embodiment of the invention, the mineral phasecomprises apatite crystals also comprising at least hydroxide ions andin which the phosphate ions (type B) and/or the hydroxide ions (type A)are partially replaced with carbonate ions.

In these hydroxyapatites, one or more sites of the crystalline structurecan be ion-free. In this case, they are nonstoichiometrichydroxyapatites comprising what is then referred to as one or more iongaps.

In another embodiment, the mineral phase comprises crystals of apatitecomprising Ca²⁺ ions, PO₄ ³⁻ ions and OFF ions, and in which at leastone of the Ca²⁺, PO₄ ³⁻ or OFF ions is partially replaced with otherions.

Among the ions capable of partially replacing the Ca²⁺ ions, mention maybe made of Mg²⁺, Cu²⁺, Sr²⁺, Ba²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Na⁺, K⁺ and Eu³⁺ions.

Among the ions capable of partially replacing the PO₄ ³⁻ ions, mentionmay be made of CO₃ ²⁻, SiO₄ ³⁻, AsO₄ ³⁻, MnO₄ ³⁻, VO₄ ³⁻, CrO₄ ³⁻ andHPO₄ ²⁻ ions.

Among the ions capable of partially replacing the OH⁻ ions, mention maybe made of CO₃ ²⁻, F⁻, Cl⁻, Br⁻, I⁻, S²⁻ and O²⁻ ions.

The material according to the invention may also contain a minute amountof proteoglycans, of glycosaminoglycans and/or of organic moleculeswhich promote mineralization. The term “minute amount” is intended tomean a proportion of less than 2%.

The characteristics of a material of the invention can be determined byoptical microscopy analyses, scanning electron microscopy SEM analyses,transmission electron microscopy TEM analyses and X-ray diffractionanalyses.

Semi-thin sections of a material according to the invention, observed bypolarized-light optical microscopy, show birefringence properties. In anideal case, the observation of alternating illuminated bands andextinguished bands associated with the movement of these fringes duringthe rotation of the microscope platform indicates a helicoidalstructure.

Samples of the material of the invention, analyzed by SEM, show orientedfibrils immerged in a mineralized layer, without individualizedcrystalline aggregates of about a micrometer.

Small-angle X-ray scattering images, taken on a material of theinvention, show the anisotropic signal of the collagen fibrils and theharmonics of the period D=67 nm. Wide-angle X-ray scattering images showthe main peaks of the apatite phase. The existence of a coalignmentbetween the signal of the fibrils and that of the mineral characterizesthe material of the invention. This coalignment is more particularlydemonstrated locally in the zones where the fibrils are aligned.

A material according to the invention can be obtained by means of amethod which consists in preparing an initial acidic aqueous solution ofcollagen which is a precursor for the organic phase (I), and at leastone aqueous solution of precursors for the mineral phase (II), and inprecipitating the collagen by increasing the pH to a value of at least7. It is characterized in that:

-   -   the concentration of collagen in the acidic aqueous solution is        at least 75 mg/ml and remains constant during said increase in        pH,    -   the mineral phase precursors comprise at least one calcium salt        and at least one phosphate salt,    -   the precipitation of the mineral phase (II) is carried out by        bringing the mineral phase precursor solution into contact with        the organic phase (I), said bringing into contact being carried        out before or after the precipitation of said organic phase (I).        The duration of the contact is set according to the speed of        precipitation and the level of mineral filler envisioned in the        final material.

In a first embodiment of the method, the collagen of the organic phase(I) is precipitated before it is brought into contact with a neutralsolution of precursors for the mineral phase (II). In this case, thesolution of precursors for the mineral phase (II) contains at least saidcalcium salts and at least said phosphate salts. The proportion ofmineral phase in the final material is modulated through the amount ofions introduced into the solution. In this embodiment, the collagenacquires its to fibrillar structure before it is brought into contactwith the mineral phase.

In a second embodiment of the method, an acidic solution of precursorsfor the organic phase (I) is brought into contact with an acidicsolution of precursors for (II). The mixture is then subjected to anincrease in pH which induces coprecipitation of the collagen and of theapatite. In this embodiment, it is is particularly important to keep theinitial concentration of collagen constant. A first means for avoidingdilution is to contain the concentrated acidic solution of precursorsfor the organic phase. (I) in a mold, the shape of which is suitable forthe desired use and which is enclosed in a dialysis membrane. A secondmeans is to introduce the concentrated collagen solution into a flexibleenvelope constituted of a dialysis membrane. The mold or said envelopeis then immersed in the solution of precursors for the mineral phase(II).

When the mineral phase II of the material in accordance with theinvention consists of crystals of pure hydroxyapatite, the method ispreferably carried out in a closed chamber in which are placed:

-   -   at least one first container containing an aqueous solution of        at least one phosphate salt and of at least one calcium salt,        which are precursors for the mineral phase II, in which solution        at least one dialysis bag is immersed, said dialysis bag        containing an initial acidic aqueous solution of collagen which        is a precursor for the organic phase (I),    -   at least one second container containing an aqueous ammonia        solution or an (NH₄)₂CO₃ powder;        it being understood that:    -   the (volume of mineral phase I precursor solution)/(closed        chamber internal volume) ratio is approximately 2×10⁻³,    -   the height of the mineral phase 1 precursor solution contained        in the first container ranges from 3 to 5 cm approximately, the        diameter of said container being from 2 to 5 cm approximately;    -   the (volume of aqueous ammonia solution)/(closed chamber        internal volume) ratio is 8×10⁻³ or the (volume of (NH₄)₂CO₃        powder)/(closed chamber internal volume) ratio is 6×10⁻³        approximately.

When these conditions are adhered to, a material in which the mineralphase II consists of pure apatite, free of any other type of calciumphosphate phase, is obtained.

The initial acidic aqueous solution of collagen preferably has thefollowing characteristics:

-   -   its collagen concentration is between 75 mg/mL and 1000 mg/mL,        preferably between 100 mg/mL and 400 mg/mL,    -   its pH is less than 4, preferably less than 3, in the presence        of acids, preferably 0.5 M acetic acid.

The solution of precursors for the mineral phase (II) preferably has thefollowing characteristics:

-   -   the concentration of calcium precursor, for example CaCl₂, is        less than the solubility limit, preferably from 2.5 mM to 1.5 M,        more particularly from 11 to 550 mM;    -   the concentration of phosphate precursor, for example NaH₂PO₄,        is less than the solubility limit, preferably from 1.5 to 900        mM, more particularly from 66 to 330 mM;    -   the amounts of precursors are such that the Ca/P molar ratio is        between 1.5 and 1.8, preferably about 1.67.

By way of example, the solubility limit at 20° C. is 7.08 M for NaH₂PO₄,and 3.83 M for CaCl₂.

When the mineral phase of the desired material comprises crystals ofapatite comprising Ca²⁺ ions, PO₄ ³⁻ ions and OH⁻ ions, and in which atleast one of the Ca²⁺, PO₄ ³⁻ or OH⁻ ions is partially replaced withother ions, the mineral phase precursor solution also contains one ormore salts, the cation of which is intended to at least partiallyreplace Ca²⁺, and/or one or more salts, the anion of which is intendedto at least partially replace PO₄ ³⁻ and/or OH⁻.

The salts of the cations intended to replace Ca²⁺ are advantageouslychosen from salts containing monovalent or divalent cations, forinstance MgCl₂, BaCl₂, SrCl₂, NaCl, KCl and NH₄Cl. The CO₃ ²⁻ precursormay be NaHCO₃. The amount of CO₃ ²⁻ precursor is preferably such thatthe NaH₂PO₄/NaHCO₃ ratio is equal to 1. In the presence of carbonate,the Ca/(P+C) molar ratio is between 1.5 and 1.8, preferably about 1.67.

The mineral phase (II) precursor solution may also containproteoglycans, glycosaminoglycans and/or organic molecules which promotemineralization, such as acidic amino acid polymer chains, preferably apoly(aspartic acid) having a chain length of between 5 and 150 aminoacid units, preferably approximately 15, and with a concentration ofbetween 0.01 μg/mL and 1.5 mg/mL, preferably 10 μg/mL.

The increase in the pH is advantageously carried out by means of a basicgaseous atmosphere, in particular an NH₃ atmosphere, or an (NH₄)₂CO₃atmosphere in one particular embodiment in which PO₄ ³⁻ or OH⁻ ispartially replaced with CO₃ ²⁻.

In one particular embodiment, the method comprises an additional stepduring which the material obtained by coprecipitation is impregnatedwith an “SBF” (“Simulated Body Fluid”) solution analogous to abiological fluid, and then the pH of the medium is adjusted to 7.4.

-   NaCl from 137 to 213 mM (for example, 213.0 mM)-   NaHCO₃ from 1.2 to 6.3 mM (for example, 6.3 mM)-   KCl from 3 to 4.5 mM (for example, 4.5 mM)-   K₂HPO₄.3H₂O from 1 to 1.5 mM (for example, 1.5 mM)-   CaCl₂ from 2.6 to 3.8 mM (for example, 3.8 mM)-   Na₂SO₃Na₂SO₄from 0.5 to 0.75 mM (for example, 0.75 mM)-   MgCl₂.6H₂O from 1.5 to 2.3 mM (for example, 2.3 mM).

The concentrations of this SBF solution represent approximately 1.5times those actually measured for a biological fluid (cf. Zhang L.-J. etal., Mater. Lett. 2004, 58, 719-722).

The pH can be adjusted to 7.4 with a mixture oftris(hydroxymethyl)aminomethane at 0.01 mol/L and HCl at 0.01 mol/L, at37° C.

The present invention is described in greater detail by means of thefollowing examples, to which it is not, however, limited.

In the examples, a collagen type I was used which was prepared fromtails of young Wistar rats, according to the following procedure. Therat tail tendons are excised in a sterile laminar flow hood, and thenwashed in a phosphate buffered saline solution containing 137 mM ofNaCl, 2.68 mM of KCl, 8.07 mM of Na₂HPO₄ and 1.47 mM of NaH₂PO₄, inorder to remove the cells and the traces of blood. The tendons are thensoaked in a 4M NaCl solution in order to remove the remaining intactcells and to precipitate a part of the high-molecular-weight proteins.After a further wash with the buffered saline solution, the tendons aredissolved in an aqueous solution containing 500 mM of acetic acid. Theresulting solution is clarified by centrifugation at 41 000 g for 2 h.The proteins other than the collagen type I are selectively precipitatedfrom a 300 mM aqueous NaCl solution, and then removed by centrifugationat 41 000 g for 3 h. The collagen is is recovered from the supernatantby precipitation from a 600 mM NaCl aqueous solution, followed bycentrifugation at 3000 g for 45 min. The resulting pellets are dissolvedin a 500 mM aqueous acetic acid solution, and then carefully dialyzed inthe same solvent in order to completely remove the NaCl.

The solutions are kept at 4° C. and centrifuged at 41000 g for 4 hbefore being used. Solutions of collagen at various concentrations areprepared by reverse dialysis against polyethylene glycol (35 kDa, Fluka)dissolved in a 500 mM aqueous acetic acid solution, up to 50% (m/v), orby slow evaporation in a laminar flow hood. The collagen concentrationof the acidic solution was determined before fibrillogenesis bydetermination of the amount of hydroxyproline. Of course, other collagensources can be used.

EXAMPLE 1

A mineral phase precursor solution was prepared by dissolving, in 40 mLof water, 110 mM of NaH₂PO₄, 66 mM of CaCl₂, 500 mM of acetic acid and0.40 μg of poly(aspartic acid). The solution is equilibrated at pH 2.2.

The collagen solution used in this example contained an amount ofcollagen of approximately 300 mg/mL. It is in the form of a partiallyfibrillar elastic gel.

The collagen solution was introduced into a dialysis bag (MW=3500 Da),and the bag was placed in the inorganic phase precursor solution in anopen container. Said container was then placed under an ammoniaatmosphere until complete precipitation of the salts at a temperature of20° C.

The ammonia atmosphere caused a coprecipitation of collagen andhydroxyapatite, which was visible from 3 hours onward. The reactionmedium can be left to mature for 8 days.

The samples were washed by immersion in a solution, advantageously a PBSphosphate buffered solution.

FIG. 2 illustrates the analysis of the material by X-ray scattering.FIG. 2 a represents an SAXS image and FIG. 2 b represents a WAXS image.The SAXS to image shows the anisotropic signal of the collagen fibrilsand the harmonics of the period D =67 nm; this demonstrates theperiodicity of the striations every 67 nm along the main axis of thefibrils. The WAXS image shows that the (002) reflection, characteristicof the presence of apatite, is reinforced in the same direction as thefibril signal observed in (a). This therefore indicates that the c axisis of the apatite crystals is oriented along the main axis of thecollagen fibrils. The signal corresponding to the interdistanced_(lateral) of the collagen molecules in the fibril is perpendicular tothe (002) reflection and parallel to the (300) reflection of theapatite. The inter-reticular planes are therefore preferentiallyoriented according to the direction of the collagen molecules. ThisX-ray scattering signature is comparable to that found on bone.

FIG. 3 represents an SEM micrograph. It shows the presence ofmineralized oriented fibrils (aligned domains).

FIG. 4 represents a semi-thin section stained with toluidine blue, whichserves as a contrast agent for the material, observed by polarized-lightoptical microscopy between crossed polarizers (A,B). 4B represents thesame zone as 4A, also observed between crossed polarizers but rotated45° relative to 4A. The birefringence is due, on the one hand, to theorganization of the organic phase and, on the other hand, to theimpregnation thereof with the mineral phase. The variation in thebirefringence bands between the two positions of the polarizersindicates the coexistence of distinct domains: (i) an aligned domaincharacterized by a zone of birefringence which is extinguished between4A and 4B since the mineralized collagen fibrils are aligned with oneanother; and (ii) a cholesteric domain characterized by a zone ofbirefringence of which the alternating of light and dark bands invertsbetween 4A and 4B since the orientation of the mineralized collagenfibrils rotates regularly from one plane to the other. A third domaincoexists with the previous two; this is an “isotropic” domain in whichthe mineralized collagen fibrils are randomly distributed in thematerial; this domain therefore exhibits no zone of birefringence in 4Aand 4B.

Measurements of the mechanical properties of the material thus preparedwere also carried out, in particular the elastic modulus, according tothe nanoindentation technique. The measurements were carried out with aUbi 1 nanomechanical indentation system (Hysitron Inc., Minneapolis,Minn., USA) and a Berkovich indenter tip, ˜10 μm². It was found that theratio of the elastic moduli at 0° and 90° relative to the longitudinalaxis of the collagen fibrils is 1.43±1.18. The 10 order of magnitude ofthis ratio is comparable to that obtained for a native compact bone,i.e. 1.50±0.315, indicating that the degree of anisotropy of the presentmaterial, and thus its fibrillar organization, is similar to that foundin bone.

EXAMPLE 2

A dilute solution of collagen (1 mg/L) was injected, in such a way as tocounter water evaporation, into a 15 μL. glass microchamber. Theinjection was continued until a dense liquid crystalline collagen phasewas obtained. The collagen was precipitated under an ammonia atmosphere,and the microchamber was then immersed in the solution of mineralprecursors (said solution containing: 213.0 mM NaCl, 6.3 mM NaHCO₃, 4.5mM KCl, 1.5 mM K₂HPO₄.3H₂O, 3.8 mM CaCl₂, 0.75 mM Na₂SO₃Na₂SO₄ and 2.3mM MgCl₂.6K₂O) adjusted to pH 7.4 and kept in this solution for a periodof 6 months at a temperature of 37° C.

The precipitated material was then washed by immersion in a phosphatebuffered solution (PBS).

FIG. 5 illustrates the analysis of the material by X-ray scattering. 5 arepresents an SAXS image and 5 b represents a WARS image. The two imagesare analogous to those of example 1.

EXAMPLE 3

A collagen solution diluted to 5 mg/mL, previously dialyzed against asolution of NaH₂PO₄ (66 mM) and CaCl₂ (110 mM), was injected, in such away as to counter water evaporation, into a 15 μL glass microchamber.The injection was continued until a dense liquid crystalline collagenphase was obtained. The microchamber was immersed in a solution ofinorganic phase precursors that was analogous to that of example 1, inan open container. Said container was then placed under an ammoniumcarbonate atmosphere until complete precipitation of the salts at atemperature of 20° C.

The atmosphere of ammonia and carbon dioxide caused a coprecipitation ofcollagen and hydroxyapatite, which was visible from 3 hours onward. Thereaction medium can be left to mature for 8 days.

The samples were washed by immersion in a phosphate buffered solutionPBS.

FIG. 6 represents an SEM micrograph. It shows the presence ofmineralized fibers exhibiting a helicoidal organization (cholestericdomain).

1. A synthetic material comprising: an organic phase (I); and a mineralphase (II), wherein the organic organic phase (1) comprises striatedcollagen fibrils constituted of collagen I triple helices and in whichthe periodicity of the striations is 67 nm, said fibrils being organizedover a large distance according to a 3D geometry associating aligneddomains and cholesteric domains, and also isotropic domains where theyare not organized; the mineral phase (II) comprises apatite crystalshaving a hexagonal crystalline structure, space group 6/m, said crystalscomprising at least calcium ions and at least phosphate ions; the axis cof the apatite crystals of the mineral phase is coaligned with thelongitudinal axis of the striated collagen fibrils of the organic phase;the collagen content in said material is at least 75 mg/cm³.
 2. Thematerial as claimed in claim I, wherein the mineral phase consists ofcrystals of pure hydroxyapatite.
 3. The material as claimed in claim 1,wherein the mineral phase consists of crystals of stoichiometrichydroxyapatite of formula (I) below:Ca₁₀(PO₄)₆(OH)₂   (I)
 4. The material as claimed in claim 3, wherein theCa/P atomic ratio of the crystals of hydroxyapatite of formula (I) is1.67.
 5. The material as claimed in claim I, wherein the mineral phasecomprises apatite crystals also comprising at least hydroxide ions andin which the phosphate ions and/or the hydroxide ions are partiallyreplaced with carbonate ions.
 6. The material as claimed in claim 1,wherein the mineral phase comprises crystals of apatite comprising Ca²⁺ions, PO₄ ³⁻ ions and OFF ions, and in which at least one of the Ca²⁺,PO₄ ³⁻ or OH⁻ ions is partially replaced with other ions, it beingunderstood that: the ions capable of partially replacing the Ca²⁺ ionsare chosen selected from the group consisting of Mg²⁺, Cu²⁺, Sr²⁺, Ba²⁺,Zn²⁺, Cd²⁺, Pb²⁺, Na⁺, K⁺ and Eu³⁺ ions; the ions capable of partiallyreplacing the PO₄ ³⁻ ions are selected from the group consisting of CO₃²⁻, SiO₄ ³⁻, AsO₄ ³⁻, MnO₄ ³⁻, VO₄ ³⁻, CrO₄ ³⁻ and HPO₄ ²⁻ ions; and theions capable of partially replacing the OH⁻ ions are selected from thegroup consisting of CO₃ ²⁻, F, Cl⁻, Br⁻, I⁻, S²⁻ and O²⁻ ions.
 7. Thematerial as claimed in claim 1, wherein said material further comprisesan amount of proteoglycans, of glycosaminoglycans and/or of organicmolecules which promote mineralization, of less than 2%.
 8. A method forpreparing a material as claimed in claim 1, which consists in preparingan initial acidic aqueous solution of collagen which is a precursor forthe organic phase (I), and at least one aqueous solution of precursorsfor the mineral phase (II), and in precipitating the collagen byincreasing the pH to a value of at least 7, wherein: the concentrationof collagen in the acidic aqueous solution is at least 75 mg/ml andremains constant during said increase in pH, the mineral phaseprecursors comprise at least one calcium salt and at least one phosphatesalt, the precipitation of the mineral phase OD is carried out bybringing the mineral phase precursor solution into contact with theorganic phase (I), said bringing into contact being carried out beforeor after the precipitation of said organic phase (I).
 9. The method asclaimed in claim 8, wherein the collagen of the organic phase (I) isprecipitated before it is brought into contact with a neutral solutionof precursors for the mineral phase (II), said neutral solutioncontaining at least said calcium salts and at least said phosphatesalts.
 10. The method as claimed in claim 8, wherein an acidic solutionof precursors for the organic phase (I) is brought into contact with anacidic solution of precursors for the mineral phase (II), and theresulting mixture is subjected to an increase in pH which inducescoprecipitation of the collagen and the apatite.
 11. The method asclaimed in claim 10, wherein the concentrated acidic solution ofprecursors for the organic phase (I) is contained in a mold which isenclosed in a dialysis membrane, the whole then being immersed in thesolution of precursors for the mineral phase (II).
 12. The method asclaimed in claim 10, wherein the concentrated solution of collagen isintroduced into a flexible envelope constituted of a dialysis membrane,said envelope then being immersed in the solution of precursors for themineral phase (II).
 13. The method as claimed in claim 8, for preparinga material in which the mineral phase II consists of crystals of purehydroxyapatite and as defined in claim 2, it wherein said method iscarried out in a closed chamber in which are placed: at least one firstcontainer containing an aqueous solution of at least one phosphate saltand at least one calcium salt, which are precursors for the mineralphase II, in which at least one dialysis bag is immersed, said dialysisbag containing an initial acidic aqueous solution of collagen which is aprecursor for the organic phase (I), at least one second containercontaining an aqueous ammonia solution or an (NH₄)₂CO₃ powder; it beingunderstood that: the (volume of mineral phase I precursorsolution)/(closed chamber internal volume) ratio is 2×10⁻³, the heightof the mineral phase 1 precursor solution contained in the firstcontainer ranges from 3 to 5 cm, the diameter of said container beingfrom 2 to 5 cm approximately; the (volume of aqueous ammoniasolution)/(closed chamber internal volume) ratio is 8×10⁻³ or the(volume of (NH₄)₂CO₃ powder)/(closed chamber internal volume) ratio is6×10⁻³.
 14. The method as claimed in claim 8, wherein the initial acidicaqueous solution of collagen has the following characteristics: itscollagen concentration is from 75 mg/mL to 1000 mg/mL; its pH is lessthan 4, in the presence of acids.
 15. The method as claimed in claim 8,wherein the solution of precursors for the mineral phase (II) has thefollowing characteristics: the concentration of calcium precursor isless than the solubility limit; the concentration of phosphate precursoris less than the solubility limit; the amounts of precursors are suchthat the Ca/P molar ratio is between 1.5 and 1.8.
 16. The method asclaimed in claim 8, for preparing a material as claimed in claim 6,wherein the solution of precursors for the mineral phase also containsone or more salts, the cation of which is intended to at least partlyreplace Ca²⁺, and/or one or more salts, the anion of which is intendedto at least partially replace PO₄ ³⁻ or OH⁻.
 17. The method as claimedin claim 8, for preparing a material as claimed in claim 7, wherein thesolution of precursors for the mineral phase (II) also containsproteoglycans, glycosaminoglycans and/or organic molecules which promotemineralization.
 18. The method as claimed in claim 8, wherein theincrease in the pH is carried out by means of a basic gaseousatmosphere.